Multipass cavity for illumination and excitation of moving objects

ABSTRACT

An illumination system for increasing a light signal from an object passing through a reflection cavity. The reflection cavity is disposed between spaced-apart, opposed first and second surfaces disposed on opposite sides of a moving stream of objects. A light collection system is disposed substantially orthogonal to a plane passing through the surfaces and the stream so as to collect light that is scattered from or emitted by the objects as they pass through a field of view disposed between the first and second surfaces. A beam of light from a laser source is directed through the stream of moving objects in a direction nearly orthogonal to the stream (but slightly inclined) and lying in the plane that extends through the surfaces and the stream. Due to the reflection angle and the distance between the stream and the first surface, the point at which the light reflected from the first surface intersects the stream on a second pass is displaced from where it passed though the stream on its initial pass. The light is reflected back and forth between the surfaces a plurality of times, illuminating a different portion of the field of view with each pass until, having ranged over the field of view, the light exits the reflection cavity. The “recycling” of the light beam in this manner substantially improves the SNR of the detection system by increasing an average illumination intensity experienced by the objects in the stream.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/490,478, filed on Jan. 24, 2000, now U.S. Pat. No.6,249,341, the benefit of the filing date of which is hereby claimedunder 35 U.S.C. §120, which claims the benefit of Provisionalapplication No. 60/117,203, filed Jan. 25, 1999.

FIELD OF THE INVENTION

This invention generally relates to illumination of moving objects orparticles for purposes of analysis and detection, and more specifically,to an apparatus and method for increasing the amount of incident lightupon these objects to increase scattered, fluorescent, and other signalsfrom moving objects, such as cells, and for detecting the presence andcomposition of Fluorescence In-Situ Hybridization (FISH) probes withincells.

BACKGROUND OF THE INVENTION

There are a number of biological and medical applications that arecurrently impractical due to limitations in cell and particle analysistechnology. Examples of such biological applications include battlefieldmonitoring of known airborne toxins, as well as the monitoring ofcultured cells to detect the presence of both known and unknown toxins.Medical applications include non-invasive prenatal genetic testing androutine cancer screening via the detection and analysis of rare cells(i.e., cells with low rates of occurrence) in peripheral blood. All ofthese applications require an analysis system with the followingprincipal characteristics:

1. the ability to carry out high-speed measurements;

2. the ability to process very large samples;

3. high spectral resolution and bandwidth;

4. good spatial resolution;

5. high sensitivity; and

6. low measurement variation.

In prenatal testing, the target cells are fetal cells that cross theplacental barrier into the mother's blood stream. In cancer screening,the target cells are sloughed into the blood stream from nascentcancerous tumors. In either case, the target cells may be present in theblood at concentrations of one to five target cells per billion bloodcells. This concentration yields only 20 to 100 cells in a typical 20 mlblood sample. In these applications, as well as others, it is imperativethat the signal derived in response to the cells be as strong aspossible to provide distinct features with which to discriminate thetarget cells from other artifacts in the sample.

It would be desirable to increase the amount of light incident uponobjects in a sample compared to prior art systems, thereby increasingthe signal-to-noise ratio (SNR) of a processing system, improvingmeasurement consistency, and thus, increasing the discriminationabilities of the system. A spectral imaging cell analysis system isdescribed in a pending commonly assigned U.S. patent application Ser.No. 09/490,478, filed on Jan. 24, 2000 now U.S. Pat. No. 6,249,341, andentitled, “Imaging And Analyzing Parameters Of Small Moving Objects SuchAs Cells,” the drawings and disclosure of which are hereby specificallyincorporated herein by reference. This previously filed applicationdescribes one approach that is applicable to imaging. It would also bedesirable to obtain many of the benefits disclosed in theabove-referenced copending application in non-imaging flow cytometersthat employ photomultiplier tube (PMT) detectors and any other systemthat relies on the illumination of objects within a cavity. Dependingupon the configuration, substantial benefits should be obtained byincreasing the amount of light incident upon an object by as much as afactor of ten or more. Such an increase in the amount of light wouldenable the use of low power continuous wave (CW) and pulsed lasers inapplications that would otherwise require the use of more expensive highpower lasers. However, if high power lasers are used for a light source,a processing system should yield higher measurement consistency, highersystem throughput, greater illumination uniformity, and other benefitsthan has been possible with prior systems.

It is a goal in the design of fluorescence instruments to achievephoton-limited performance. When photon-limited performance is achieved,noise sources in the instrument are reduced to insignificance relativeto the inherent statistical variation of photon arrivals at thedetector. A good example of photon-limited design is found innon-imaging flow cytometers. The PMT detectors employed in theseinstruments can amplify individual photons thousands of times with veryfast rise times.

Non-imaging cytometers take advantage of the PMT's characteristics toachieve photon-limited performance by making the illuminated area assmall as possible. Decreasing the laser spot size reduces the amount oftime required for an object to traverse a field of view (FOV) of thedetectors. The reduced measurement time, in turn, reduces the integratedsystem noise, but does not reduce the signal strength of the object. Thesignal strength remains constant because the reduced signal integrationtime is balanced by the increased laser intensity in the smaller spot.For example, if the FOV in the axis parallel to flow is decreased by afactor of two, an object's exposure time will decrease by a factor oftwo, but the intensity at any point in that FOV will double, so theintegrated photon exposure will remain constant.

The reduced noise and constant signal strength associated with a reducedFOV increases the SNR of the non-imaging cytometer up to a point. Beyondthat point, further reductions in the FOV will fail to improve the SNRbecause the dominant source of variation in the signal becomes theinherently stochastic nature of the signal. Photonic signals behaveaccording to Poisson statistics, implying that the variance of thesignal is equal to the mean number of photons. Once photon-limitedperformance is achieved in an instrument, the only way to significantlyimprove performance is to increase the number of photons that reach thedetector.

A common figure of merit used in flow cytometry is the coefficient ofvariation (CV), which equals the standard deviation of the signal overmany measurements divided by the mean of the signal. Photon noise, asmeasured by the CV, increases as the mean number of photons decreases.For example, if the mean number of photons in a measurement period isfour, the standard deviation will be two photons and the CV will be 50%.If the mean number of photons drops to one, the standard deviation willbe one and the CV will be 100%. Therefore, to improve (i.e., decrease)the CV, the mean number of photons detected during the measurementinterval must be increased. One way to increase the number of photonsstriking the detector is to increase photon collection efficiency. If anincrease in photon collection efficiency is not possible, an alternativeis to increase the number of photons emitted from the object during themeasurement interval. Accordingly, it would be beneficial to provide asystem in which illumination light incident on an object but notabsorbed or scattered is recycled and redirected to strike the objectmultiple times, thereby increasing photon emission from the object.

In the case of a conventional imaging flow cytometer, such as thatdisclosed in U.S. Pat. No. 5,644,388, a frame-based charge-coupleddevice (CCD) detector is used for signal detection as opposed to a PMT.In this system, the field of view along the axis of flow isapproximately ten times greater than that in PMT-based flow cytometers.In order to illuminate the larger field of view, the patent discloses acommonly used method of illumination in flow cytometry, in which theincident light is directed at the stream of particles in a directionorthogonal to the optic axis of the light collection system. The methoddisclosed in the patent differs slightly from conventional illuminationin that a highly elliptical laser spot is used, with the longer axis ofthe ellipse oriented along the axis of flow. As a result of thisconfiguration, the entire FOV can be illuminated with laser light. Giventhat a laser is used, the intensity profile across the illuminatedregion has a Gaussian profile along the axis of flow. Therefore, objectsat either end of the field of view will have a lower intensity ofillumination light. Unlike a non-imaging flow cytometer, the lightcollection process disclosed in this patent does not continue for theduration of the full traversal of the FOV. Instead, light is collectedfrom objects at specific regions within the FOV. Object movement duringthe light collection process is limited to less than one pixel by use ofa shutter or pulsed illumination source. As a result, the amount oflight collected from an object varies as a function of its position inthe field of view, thereby increasing measurement variability. In orderto partially mitigate this variation, the illumination spot may be sizedso that it substantially overfills the FOV to use an area of theGaussian distribution near the peak where the intensity variation isminimized. However, this approach has the undesired effect of reducingthe overall intensity of illumination, or photon flux, by spreading thesame amount of laser energy over a significantly larger area. The endresult of reducing photon flux is a reduction in the SNR.

Accordingly, it will be apparent that an improved technique is desiredto improve the SNR and measurement consistency of an instrument byincreasing photon emission from the object and improving the uniformityof illumination. It is expected that such a technique will also haveapplications outside of cell analysis systems and can be implemented indifferent configurations to meet the specific requirements of disparateapplications of the technology.

SUMMARY OF THE INVENTION

The present invention is directed to an illumination system that isadapted to increase the amount of signal emitted from an object toincrease the SNR and to improve the measurement consistency of devicesin which the present invention is applied. In general, there is relativemovement between the object and the illumination system, and although itis contemplated that either (or both) may be in motion, the object willpreferably move while the illumination system is fixed. In addition, itshould also be understood that while this discussion and the claims thatfollow recite “an object,” it is clearly contemplated that the presentinvention is preferably intended to be used with a plurality of objectsand is particularly useful in connection with illuminating a stream ofobjects.

The present invention increases the amount of light incident upon anobject as the object traverses a field of view, without incurring theexpense of additional or more powerful light sources. It also may beconfigured to increase the uniformity of illumination in the field ofview.

In a first embodiment of the present invention, a reflection cavity isformed by the placement of two mirrors on either side of a moving streamof objects. A light collection system is disposed substantiallyorthogonal to a plane extending through the mirrors and the stream. Thelight collection system is configured to collect light over a predefinedangle and within a predefined region or field of view between themirrors. Accordingly, the light collection system collects light that isscattered or emitted from objects as they traverse the space between themirrors. The scattered or emitted light that is collected is directedonto a detector.

A light from a light source is directed through the stream of movingobjects in a direction nearly orthogonal to the stream of objects butslightly inclined in the plane that extends through the mirrors and thestream. With cells and most other objects, only a small fraction of theincident light interacts with the objects via absorbance or scatter. Therest of the light passes through the stream, and is then redirected byreflection from a surface back into the stream of moving objects. Thelight leaves the reflecting surface at a reflected angle that is equalto an incident angle of the light. Due to the reflection angle and thedistance between the stream and the first surface, the light intersectsthe stream on the second pass at a position that is displaced from thatat which the light passed though the stream on its initial pass. Thelight continues through the stream and is redirected by a second surfaceon the other side of the stream, which is substantially parallel to thefirst surface, back towards the stream. Again, as a result of thereflection angle and the distance between the second surface and thestream, the light passes through the stream on the third pass at aposition that is displaced from that of the second pass. The reflectionof the light through the stream continues a plurality of times until thelight has traversed a distance along the direction in which the streamis flowing that is substantially equal to the collected field of view ofthe light collection system. At this point, the light is no longerreflected back through the stream, but is preferably caused to exit theillumination system.

It should be understood that most of the light that passes through thestream is largely unimpeded by the stream or objects in the stream.Therefore, upon subsequent passes, substantial light remains tointercept the object or objects in the stream. By “recycling” light inthis manner, the light that would normally be wasted is employed toilluminate the object each time the object passes through the light.Consequently, the SNR of the instrument is substantially improved byincreasing the amount of scattered and/or emitted light that is incidenton the detector.

The present invention can also be configured so as to obtain a desiredillumination profile along the axis of flow. The beam size and traversaldistance can be adjusted to create a predefined amount of overlapbetween beams at the stream intersection point to homogenize theintensity profile along the axis of flow. Further, the input beam can beapertured to use a section of the beam with less variation to furtherincrease illumination uniformity along the flow axis.

In a second embodiment of the invention, the beam is reflected back uponitself after numerous traversals of the cavity. In this embodiment, thetotal number of traversals is doubled relative to the first embodiment,thereby increasing the number of photons incident upon the stream.

In a third embodiment, a slight angle is introduced between the mirrorswhich causes the angle of incidence to gradually decrease as the beamtraverses the cavity. Eventually the traversal direction reverses,causing the beam to traverse the cavity in the opposite direction,thereby increasing the number of times the beam traverses the stream.

In a fourth embodiment, the present invention can be configured suchthat the surfaces which redirect the beam back into the stream containoptical power in one or both axes in order to create one or moretraversal reversals of the beam a to optimally size the beam at or nearthe intersection points with the stream.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is an isometric view of an illumination system corresponding to afirst embodiment of the present invention;

FIG. 2 (Prior Art) is an isometric view of a conventional method forilluminating objects in a flow stream;

FIG. 3 is an isometric view of an exemplary imaging system thatimplements the illumination system of FIG. 1;

FIG. 4 is an isometric view of an embodiment of the present inventionusing mirrors immersed in a fluid;

FIG. 5 is an XY plot showing an illumination intensity profile for aconventional (Prior Art) single pass illumination scheme with a shortFOV;

FIG. 6 is an XY plot showing an illumination intensity profile for aconventional (Prior Art) single pass illumination scheme with a tallFOV;

FIG. 7 is a schematic diagram illustrating a condition in which no beamoverlap occurs at the center of the cavity when a smaller beam size isused in a preferred embodiment of the present invention;

FIG. 8 is an XY plot showing an illumination intensity profile for theembodiment of the present invention illustrated in FIG. 7, but for thecase in which a narrow light beam passes through a field of view fivetimes;

FIG. 9 is a schematic diagram illustrating a condition in whichsignificant beam overlap occurs at the center of the cavity when alarger beam size is used in a preferred embodiment of the presentinvention;

FIG. 10 is an XY plot showing an illumination intensity profile for theembodiment of the present invention illustrated in FIG. 9, but for thecase in which a wide light beam passes through a field of view fivetimes;

FIG. 11 is a plot of the beam size in the horizontal axis for two beamswith different waist sizes in a five pass embodiment of the invention,illustrating how a larger waist size can produce a smaller average beamsize, thereby increasing the overall intensity of light incident on thestream;

FIG. 12 is an isometric view of an embodiment of the present inventionthat employs a retro-reflector to reverse beam traversal, increasing thenumber of times the beam traverses the flow stream;

FIGS. 13A-13F schematically illustrate an embodiment of the presentinvention where the beam traversal direction is reversed after aplurality of passes across the cavity by introducing an angle betweenthe cavity mirrors;

FIGS. 14A-14B further illustrate schematically an embodiment of thepresent invention wherein the beam traversal direction is reversed aftera plurality of passes across the cavity by introducing an angle betweenthe cavity mirrors and in which the mirrors provide an optical powerabout an axis parallel to the flow axis for refocusing the beam in thehorizontal axis with each pass through the cavity;

FIG. 15 is a plot of the beam size in the horizontal and vertical axesfor each pass of the beam across the cavity in an embodiment employing28 passes, as illustrated in FIGS. 14A-14B;

FIG. 16 schematically illustrates an embodiment of the present inventionwherein the cavity mirrors have a toroidal surface profile and providean optical power in both the horizontal and vertical axes for inducing areversal of beam traversal direction and for refocusing the beam in bothaxes; and

FIG. 17 is a plot of the beam size in the horizontal and vertical axesfor each pass of the beam across the cavity in an embodiment employing21 passes of the beam where the walls of the cavity have the toroidalsurface profile illustrated in FIG. 16.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention offers considerable advantages over the prior artfor illumination of cells and other types of particles in a flow stream.These advantages arise from the recycling of laser light to increase thephoton flux incident upon objects in a flow stream. The presentinvention can also be configured to improve the uniformity ofillumination, while at the same time increasing the photon flux incidentupon objects, which is expected to enhance the performance of variousflow cytometry applications.

A first preferred embodiment of an illumination system 10 in accord withthe present invention is shown in FIG. 1. Illumination system 10includes a rectangular solid glass substrate 14 with reflective coatings15 and 16 applied to two substantially parallel and flat outer surfaces15 a and 16 a of the glass substrate. A channel 20 is disposed in therectangular solid to enable a plurality of objects 24 in a flow streamto pass through illumination system 10 between surfaces 15 a and 16 a.As is commonly done, the objects may be entrained in a sheath flow (notshown) in order to keep them centered within channel 20. A substantiallycylindrical beam of light 12, such as that emitted by a laser source(not shown), is directed toward an uncoated area 13 in surface 15 a ofthe substrate such that a propagation axis of the beam of light(indicated by the arrow) is at a slight angle with respect to a normalto surface 15 a. The beam proceeds through surface 15 a and passesthrough at least a portion of the plurality of objects 24 and is thenreflected from reflective coating 16 back into the plurality of objects24. The angle of propagation axis 12 a is set such that as beam of light12 traverses the substrate, it rises a predefined amount, intersectingsurface 15 a in reflective coating 15 above uncoated area 13. The beamreflects from reflective coating 15 and again passes through theplurality of objects 24.

As objects 24 flow along the channel, corresponding images of theobjects are produced with an optical system (not shown in this Figure)having a field of view 25. As shown in FIG. 1, light beam 12 continuesto traverse substrate 14 such that it passes through the substrate tentimes, thereby illuminating all of field of view 25, before it isallowed to pass out of the substrate through an uncoated area 26 insurface 15 a. Reflection spots 28 and dashed lines 27 illustrate thepath of the light beam and indicate the points where the beam intersectsand reflects from reflective coatings 15 and 16. Reflective coatings 15and 16 form a reflection cavity 17 through which the plurality ofobjects 24 pass. Those skilled in the art will appreciate that surfaces15 and 16 could be independently mounted on their own substrates withoutthe use of the glass substrate 14. By reflecting the light back andforth in this manner, the total amount of light incident on objects 24is substantially increased over that provided by conventionalillumination methods.

In contrast to the foregoing configuration, FIG. 2 illustrates a commonapproach used in the prior art to illuminate objects in flow cytometerssuch as those described in U.S. Pat. No. 5,644,388. In thisconfiguration, an elliptical-shaped beam of light 30 is directed throughthe substrate 36 and passes through the plurality of objects 24. Inorder to illuminate all of field of view 38, the light beam size in theflow axis is made substantially larger than that used in the presentinvention. As a result, the intensity of light at any point in field ofview 38 is substantially less than in the present invention, whichreduces the amount of light scattered or otherwise emitted from theplurality of objects 24, thereby reducing the SNR of the conventionalapproaches relative to the SNR of the present invention. Likewise, inthe conventional approach, the illumination intensity varies across thefield of view in accordance with a Gaussian intensity distribution ofthe illuminating laser light.

FIG. 3 shows an exemplary imaging system 40 that is substantiallysimilar to imaging systems disclosed in copending commonly assigned U.S.patent application Ser. No. 09/490,478, the specification and drawingsof which have been specifically incorporated herein by reference. Thepresent invention is employed for illumination in imaging system 40. Inthis imaging system, light 41 from an object passes through a collectionlens 42, which collects the light, producing collected light 43. Thecollected light is focussed substantially at infinity, i.e., the rays ofcollected light 43 are generally parallel and enter a prism 44, whichdisperses the light, producing dispersed light 45. The dispersed lightenters an imaging lens 46, which focusses light 47 on atime-delay-integration (TDI) detector 48.

Imaging system 40 includes illumination system 10, which was discussedabove. A laser light source 50 directs a beam of coherent light 51toward a reflection cavity 52 within illumination system 10, as shown inthe Figure. Optionally, the illumination system may further include anaperture plate 53, which includes an aperture 53 a having a diameterselected to reduce the size of the beam sufficiently so that the lightintensity distribution across the cross section of the beam that haspassed through the aperture is substantially constant. It should benoted that the present invention may be included in other imagingsystems that are described and illustrated in the above referencedcopending patent application.

The present invention can also be configured for implementation in astereoscopic imaging flow cytometer. This configuration of the presentinvention is shown in FIG. 4 where a reflection cavity 59 is created bysupporting two mirrors 55 and 56 on independent substrates within animmersion medium of an imaging flow cytometer. The ends of two capillarytubes 64 a and 64 b are brought within close proximity to each other. Astream of objects 63 is hydrodynamically focused with capillary tube 64a and caused to flow through a gap 64 c between the tubes and intocapillary tube 64 b. Two water immersion objectives 61 and 66 aremounted on a frame (not shown) and are employed to image the gap betweenthe capillary tubes onto two pixilated detectors 62 and 67. Mirrors 55and 56, which are supported within the immersion cavity on a frame 60,create reflection cavity 59 around the stream of objects 63. Light froman illumination source (not shown) is directed along a path 65 undermirror 55, through stream of objects 63, and onto mirror 56. Uponstriking the mirror, the light is redirected back through stream ofobjects 63, and caused to again traverse the stream of objects,generally in the manner described above, in regard to FIG. 1.

The foregoing Figures illustrate several of the various optical systemconfigurations that include the present invention. Those skilled in theart will appreciate the present invention may be used to advantage inimaging as well as non-imaging flow cytometers. The following discussionnumerically quantifies the advantage of using an embodiment of thepresent invention in a non-imaging PMT-based flow cytometer. The signalstrengths are compared for three different illumination systems, two ofwhich are in the prior art, and one of which is an embodiment of thepresent invention.

The first prior art system to be discussed is incorporated in awidely-available, non-imaging commercial flow cytometer system. Thissystem employs a 15 mW continuous wave laser that produces an ellipticalbeam spot 70 microns wide by 20 microns tall, a 6 m/s sample flow rate,and a PMT detector (not shown). An intensity profile along a flow pathof the illumination system is illustrated in FIG. 5. The profile has apeak intensity 107 that is approximately 0.68 photons/microsecondthrough the area defined by the absorbance cross section of afluorescein molecule. The intensity varies over the field of view of thecollection system in accordance with a Gaussian distribution function,1/e^(2x), wherein “x” is a ratio of the distance along the traversalpath to the radius of the beam. Conventionally, the boundaries of aGaussian beam are defined at a 1/e² point 108, which is the position atwhich the intensity falls to approximately 13% of the peak intensity.For this illumination profile, each fluorescein molecule emits anaverage of 1.29 photons as it traverses the illuminated region. Thoseskilled in the art will appreciate that the emission of photons isquantized (no fractional photons are emitted) and that some moleculesemit no photons, while others emit one or more photons when traversingthe illuminated region. However, the resulting average number ofemissions per molecule over all molecules is a fractional number.

The second prior art example is the same as the first except that thedimension of the illuminating beam is 500 microns in the axis parallelto the direction of object flow. FIG. 6 illustrates an intensity profilefor the enlarged illumination area produced by this second prior artsystem. A peak intensity 109 for this profile is approximately 0.027photons/microsecond, which is 25 times lower than in the first exampleshown in FIG. 5. Despite the lower peak intensity, the average emissionper fluorescein molecule remains 1.29 photons due to the increasedillumination time allowed by the taller beam. Because there is nodifference in the average emission per fluorescein dye molecule in thetwo prior art systems, there is no change in instrument performance,despite the 25-fold change in beam height. Changes in the beam heightalong the axis of the flow stream do not change the number offluorescent photons emitted by the sample as it flows through theilluminated region, because the increased illumination time is offset bya corresponding reduced photon flux per unit area.

FIG. 7 illustrates an embodiment of the present invention wherein thebeam height is 100 microns in the axis parallel to flow, and the beam isreflected across the illuminated region five times. The beam incidentangle is inclined relative to the reflecting surfaces so that there isno overlap of the beam in the center of the cavity. The resulting totalilluminated height is therefore 500 microns, like that of the secondprior art example discussed just above. In this embodiment of thepresent invention, the beam width is increased from the 70 microndimension in the prior art, to 90 microns in order to reduce beamdivergence. With the configuration used in this embodiment of thepresent invention, the average number of photons emitted per dyemolecule is increased to 4.78 photons, more than a factor of threegreater than is obtained using conventional illumination in the priorart. The increase in emitted photons is a result of two factors: (1)high illumination flux due to compact beam dimensions; and (2) anextended illumination height (and correspondingly longer illuminationtime), due to the multiple offset passes of the laser beam through theillumination region.

The intensity profile along the stream axis, which provides theincreased illumination flux of the above embodiment, is illustrated inFIG. 8. From FIG. 8, it is apparent that a five-pass embodiment producesa peak intensity 138 of more than 0.10 photons/microsecond through thearea defined by the absorbance cross section of the fluoresceinmolecule, which is four times greater than that shown in FIG. 6 for theprior art illumination configuration with the same illumination height.The increase in intensity of the exciting beam and the increase in thenumber of passes in which the exciting beam encounters a molecule in thepresent invention produce more fluorescence from each molecule of thedye.

In addition to increasing illumination intensity, the present inventionenables control of the illumination intensity profile in the cavity atthe intersection of the stream and each of the plurality of beam passes.By appropriately choosing a waist size and the incident angles, anadvantageous illumination profile may be achieved. For applications ofthe present invention in imaging flow cytometers, it may be advantageousto create a more uniform illumination intensity profile in the cavity,to decrease measurement variation. FIG. 9 shows a configuration similarto that of FIG. 7, except that the beam height is increased to produce a50% overlap between beam segments in adjacent passes. FIG. 10 shows theresulting intensity profile, which is of much higher uniformity than isproduced under no-overlap conditions. In addition to changing the beamsize, the extent of beam overlap from pass to pass can be controlled bymodifying the distance between the cavity's reflective surfaces and bychanging the incident angle of the beam. Increasing the distance betweenthe reflective surfaces and/or the incident angle enables the beam topropagate farther along the vertical axis between passes across thecenter of the cavity.

In addition to the factors that cause beam overlap noted above, beamoverlap can occur as a result of a divergence of the beam as ittraverses the cavity. Divergence due to diffraction causes thecross-sectional area of the beam to increase as the beam traverses thecavity. As the traversal distance increases, a concomitant increase incross-sectional beam area, or beam spread occurs. This increase in beamspread decreases the intensity, or photon flux at any given portion inthe cross section of the beam, which in turn, reduces the probability offluorescence excitation of probe molecules. Therefore, the beam spreadmust be kept within acceptable limits. In accord with the embodiments ofthe present invention discussed above, the beam waist, i.e., the pointof the smallest cross-sectional area of the beam, is preferably at amidpoint of the beam traversal through the cavity. The beamcross-sectional size increases in either direction away from the waistat a rate that is inversely proportional to the size of the waist. Thisphenomenon is illustrated in FIG. 11, which shows the spread of twobeams over five passes across the center of a 5 mm wide cavity, one beamhaving a 50 micron waist (line 141 with triangles at data points) andthe other an 80 micron waist (line 143 with squares at data points).Even though a 50 micron waist is substantially smaller in diameter thanan 80 micron waist, the average beam diameter throughout the entiretraversal of the 50 micron beam is larger. Those skilled in the art willappreciate that in view of the beam divergence, the waist size may bechosen appropriately to maximize intensity based on the number of cavitytraversals and the acceptable beam size at points away from the waist,or in regard to the average beam size within the cavity. Those skilledin the art will also appreciate that the beam waist may be disposedappropriately within or outside the cavity to achieve a desired effectwith the present invention.

Within the scope of the present invention, various parameters can beadjusted to increase the number of cavity traversals the beam makeswhile maintaining a beam size that is appropriate to increasefluorescence. For example, the cavity may be made narrower to decreasethe path length that the beam must travel as it traverses the cavity. Inthis manner, the number of passes in the cavity can be increased whilestill maintaining a small cross-sectional beam size and therebymaintaining relatively high beam intensity.

FIG. 12 shows an embodiment of an illumination system 10′ in accord withthe present invention in which a retro-reflector 139 is included toreflect the beam back into the cavity after it has exited the top of thecavity. In all other respects, the embodiment shown in this Figure issubstantially identical to the first preferred embodiment shown in FIG.1. However, in the embodiment of FIG. 12, retro-reflector 139 reflectsthe beam back along the path it followed before exiting the cavity, sothat the beam reversing its previous path through the substrate. Thisembodiment effectively doubles the number of beam passes through thecavity achieved by the embodiment in FIG. 1. A 20-pass retro-reflectedembodiment will provide nearly a 15-fold increase in the average photonexposure of an object over a conventional single-pass illumination.

FIGS. 13A-13F illustrate another embodiment of the present inventionwherein the beam traverses the cavity and reverses direction, but unlikethe embodiment of FIG. 12, it does so without the use of aretro-reflector. In this embodiment, an angle is introduced between twosurfaces 15 a′ and 16 a′, which comprise the walls of the cavity. Forillustration purposes, the angle between the reflecting surfaces isexaggerated and shown as equal to ten degrees. As will be observed inthe Figure, the introduction of the angle between the two surfacescauses a gradual reduction in the incident angle of the beam relative tothe surfaces, as the beam repeatedly traverses the cavity. Eventually,the incident angle becomes 90 degrees, or reverses sign, and the beam isreflected back upon itself (or down the walls of the cavity) andre-traverses the cavity in the opposite direction.

As illustrated earlier in FIG. 11, the cross-sectional beam sizeconverges to a minimum at the waist position then diverges. Theintensity of the beam at any point is inversely proportional to thesquare of the beam diameter. In order to maintain a high beam intensity,it is therefore advantageous to maintain a small beam diameter as thebeam traverses the cavity. To achieve this goal, the embodiment of thepresent invention shown in FIGS. 14A and 14B incorporates optical powerin the reflecting surfaces of the cavity. Each wall of the cavity is acylindrical mirror 151 and 153 with curvature in the horizontal planeselected to focus the light beam that is reflected therefrom within thecavity. The center of each wall's radius of curvature, R, is the flowstream, so with each reflection of the light beam, the diverging beam isrefocused by the mirrors on the objects within the flow stream. As aresult, a small beam diameter is maintained in the vicinity of the flowstream, and the beam spread in the axis perpendicular to flow isminimized so that more light is focused on the objects in the streamthan could otherwise be obtained. The embodiment shown in FIGS. 14A and14B also incorporates the method of beam reversal illustrated in FIG.13. The size of a 488 nm laser beam waist in the vertical and horizontalaxes for this embodiment is plotted in FIG. 15. The beam size in theaxis perpendicular to flow is maintained at 40 microns in the vicinityof the flow stream. As the beam propagates up the cavity, the beamdiameter alternately converges on the flow stream and then divergestoward the reflecting surface where, upon reflection, the beamre-converges near the flow stream. In this embodiment of the presentinvention, the cylindrical surface contains no optical power in the axisof flow. Therefore, the beam diameter upon the first intersection withthe stream is 199 microns. The beam continues to converge up to the14^(th) pass where the beam waist, or minimum beam diameter, of 91microns is reached. The use of the tilted surface wall or the use of aretro-reflector where the beam exits enables the beam to traverse backdown the cavity, providing a total of 28 passes of the beam through theflow stream. A flow cytometer employing this embodiment, with a 28-passcavity, produces an average photon emission per dye molecule of 44.32photons. This result represents a 35-fold increase in signal strengthcompared to the conventional method of illumination, where only a 1.29photon per molecule average emission is achieved.

As a further embodiment of the present invention, optical power can beprovided in both the vertical and horizontal axes of the cavity walls.FIG. 16 illustrates an alternative embodiment of the present inventionwhere the reflection cavity surfaces 161 and 163 are toroids with aradius of 50 mm about an axis perpendicular to the page and a radius ofapproximately 1 mm about the axis along the flow stream. The centers ofcurvature for the 50 mm surfaces are separated by approximately 98 mm,so that the vertex of each mirror is separated by 2 mm and centered onthe flow stream axis. The illumination beam enters the reflective cavityperpendicular to the flow stream axis at a point approximately 1 mmbelow the axis defined by a line running between the centers ofcurvature for the two 50 mm surfaces. Along the axis of beampropagation, the beam waist is located within the reflective cavity andthe beam makes a first flow stream intersection. The beam traverses thecavity and is reflected upward at an angle of approximately 2.3 degreesfrom horizontal, causing the beam to re-cross the cavity and strike theother wall of the cavity. The beam reflects from this cavity wall at anangle of about 4.4 degrees with respect to horizontal and continues tore-cross the cavity and strike the opposite surface in this manner suchthat the reflected angle with the horizontal increases upon eachreflection of the beam by one of the surfaces. After the sixthreflection, the beam traverses the cavity and crosses the axis definedby a line 165 running between the centers of curvature of the two 50 mmradii surfaces 161 and 163. At this point, the normals to these surfacespoint downward. Therefore, the reflected angle of the beam with respectto the horizontal decreases. At the first reflection after the beamcrosses the axis defined by the centers of curvature of the surfaces,the reflection angle of the beam with respect to the horizontal isapproximately 8.1 degrees. At the second reflection after crossing theaxis, the reflection angle is reduced to approximately 7.4 degrees. Atthe eleventh reflection, the beam makes an angle of approximately zerodegrees to the horizontal, and after striking the other wall, thepropagation direction of the beam with respect to the flow axis isreversed. The beam then propagates down the cavity, reflecting from thesurfaces and eventually exits the cavity at its point of entry aftermaking twenty two passes through the flow stream.

FIG. 17 illustrates the beam waist size during the propagation of a 488nm laser beam through the embodiment of the invention illustrated inFIG. 16, where the reflecting surfaces have optical power in both axes.The beam intersects the flow stream on the first pass with a 50 micronwaist in each axis. After the beam passes through the stream it beginsto diverge and strikes the far wall of the cavity. Upon reflection, thebeam re-converges in the vertical plane such that the waist isapproximately 50 microns when it crosses the flow stream. As describedin the previous embodiment the beam always re-converges at the flowstream with a waist size of 50 microns in the vertical plane afterstriking the cavity wall. However, in the axis parallel to flow the beamcontinues to diverge after reflecting off the cavity wall. The opticalpower in that axis is insufficient to cause the beam to re-converge.Therefore, when the beam intersects the flow axis on the second pass, itis approximately 55 microns in the axis parallel to flow. The opticalpower in the axis parallel to flow reduces the divergence from what itwould be if the surface contained no optical power in the that axis, butthe divergence continues to increase as the beam enters the far fieldpropagation regime. Ultimately, after reflecting from the left hand andright hand surfaces of the cavity eleven times, the beam begins tore-converge in the axis parallel to flow. At this point the beam waistis approximately 176 microns. From this point on the beam begins toconverge back toward a 50 micron waist, but exits the cavity beforereaching a dimension of 50 microns in the axis parallel to flow.

Those skilled in the art will appreciate that in all the cases describedthus far, the F-number of each of the optical systems described is inexcess of 40 and therefore, from an aberration perspective, the opticalperformance is very well behaved, and the spot sizes of the beam in eachaxis are dictated by diffraction theory. Therefore, constant radiussurfaces may employed. However, those skilled in the art will alsoappreciate that for lower F-numbers, or smaller spot sizes, aspheric ornon-constant radii surfaces may be employed to control wave frontaberrations.

Although the present invention has been described in connection with thepreferred form of practicing it and modifications thereto, those ofordinary skill in the art will understand that many other modificationscan be made to the present invention within the scope of the claims thatfollow. Accordingly, it is not intended that the scope of the inventionin any way be limited by the above description, but instead bedetermined entirely by reference to the claims that follow.

The invention in which an exclusive right is claimed is defined by thefollowing:
 1. An illumination system adapted to increase an amount oflight incident upon an object that is moving relative to theillumination system, comprising: (a) a light source producing a beam oflight; (b) a first reflecting surface and a second reflecting surfacedisposed opposite each other and maintained in a facing relationship soas to define a reflection cavity, the first reflecting surface and thesecond reflecting surface being separated by a defined distance, saidreflection cavity having a field of view through which the object passesbetween the first reflecting surface and the second reflecting surface,said beam of light being incident upon the first reflecting surface atan acute angle relative to a normal to the first reflecting surface, theangle and the distance being selected such that said beam of light isreflected back and forth between the first and second reflectingsurfaces so as to cross the field of view a predefined number of times,said beam of light thus being incident on the object a plurality oftimes as the object traverses the field of view; and (c) a detectordisposed such that light from the object is incident upon the detector,while said beam of light is not incident upon the detector.
 2. Theillumination system of claim 1, wherein the light source comprises alaser.
 3. The illumination system of claim 2, wherein the lasercomprises one of a continuous wave laser and a pulsed laser.
 4. Theillumination system of claim 1, wherein the object is entrained in afluid that flows through the field of view.
 5. The illumination systemof claim 1, wherein the first and second reflecting surfaces comprisefirst and second reflective coatings disposed on a transparentsubstrate.
 6. The illumination system of claim 1, wherein the angle atwhich the beam of light is incident on the first reflecting surface andthe distance are selected such that the beam of light is caused tooverlap itself as it intersects a path along which the object traversesthe field of view, when the beam is reflected between the firstreflecting surface and the second reflecting surface.
 7. Theillumination system of claim 1, wherein the angle at which the beam oflight is incident on the first reflecting surface and the distance areselected such that the beam of light passes through the field of viewwithout overlapping itself as it intersects a path along which theobject traverses the field of view, when the beam is reflected betweenthe first reflecting surface and the second reflecting surface.
 8. Theillumination system of claim 1, further including an aperture that isdisposed along a path of the beam of light so as to reduce a crosssection of the beam of light transmitted through the aperture such thata variation in light intensity across the beam of light that istransmitted through the aperture is reduced.
 9. The illumination systemof claim 1, wherein the first reflecting surface forms an acute non zeroangle with the second reflecting surface, said acute non zero anglebeing selected so that the beam of light that is reflected back andforth between successively different points along the first reflectingsurface and the second reflecting surface in a first directioneventually begins to reflect back and forth between successivelydifferent points along the first reflecting surface and the secondreflecting surface in a second direction that is opposite to the first.10. The illumination system of claim 1, wherein at least one of thefirst reflecting surface and the second reflecting surface is curved tofocus the beam of light onto an axis along which the object movesthrough the reflection cavity, to reduce a spread of the beam of lightwhere the beam of light illuminates the object.
 11. The illuminationsystem of claim 10, wherein said at least one of the first and thesecond reflecting surfaces is curved about one of a first axis that isgenerally aligned with a direction of travel of the object, and a secondaxis that is generally orthogonal to the direction of travel of theobject through the reflection cavity.
 12. The illumination system ofclaim 10, wherein said at least one of the first and the secondreflecting surfaces is curved about both a first axis that is generallyaligned with a direction of travel of the object, and a second axis thatis generally orthogonal to the direction of travel of the object throughthe reflection cavity.
 13. An illumination system adapted to increase anamount of light incident upon an object that is moving relative to theillumination system, comprising: (a) a light source producing a beam oflight; and (b) a first reflecting surface and a second reflectingsurface separated by a defined distance and disposed opposite each otherand maintained in a facing relationship so as to define a reflectioncavity, said reflection cavity having a field of view through which theobject passes between the first reflecting surface and the secondreflecting surface, said first reflecting surface being disposed in anon parallel orientation with respect to said second reflecting surface,said beam of light being incident upon the first reflecting surface atan acute angle relative to a normal to the first reflecting surface, thedistance and the angle being selected so that said beam of light isreflected back and forth between the first and second surfaces so as tocross the field of view a defined number of times, said beam of lightbeing incident on the object a plurality of times as the objecttraverses the field of view.
 14. An illumination system adapted toincrease an amount of light incident upon an object that is movingrelative to the illumination system, comprising: (a) a light sourceproducing a beam of light; and (b) a first reflecting surface and asecond reflecting surface disposed opposite each other and separated bya defined distance, such that they are maintained in a facingrelationship so as to define a reflection cavity having a field of viewthrough which the object passes between the first reflecting surface andthe second reflecting surface, at least one of said first and saidsecond reflecting surfaces being curved so as to have an optical powergreater than one, said beam of light being incident upon the firstreflecting surface at an acute angle relative to a normal to the firstreflecting surface, the angle and the distance being selected such thatsaid beam of light is reflected back and forth between the first andsecond surfaces so as to cross the field of view a specified number oftimes, said beam of light being incident on the object as the objecttraverses the field of view and focused upon the object by said at leastone of said first and second reflecting surfaces that is curved.
 15. Theillumination system of claim 14, wherein said at least one of the firstand the second reflecting surfaces is curved about one of a first axisthat is generally aligned with a direction of travel of the object, anda second axis that is generally orthogonal to the direction of travel ofthe object through the reflection cavity.
 16. The illumination system ofclaim 14, wherein said at least one of the first and the secondreflecting surfaces is curved about both a first axis that is generallyaligned with a direction of travel of the object, and a second axis thatis generally orthogonal to the direction of travel of the object throughthe reflection cavity.
 17. An illumination system adapted to increase anamount of light incident upon an object that is moving relative to theillumination system, comprising: (a) a light source producing a beam oflight; (b) a first reflecting surface and a second reflecting surfaceseparated by a defined distance and disposed opposite each other in afacing relationship so as to define a reflection cavity, said reflectioncavity having a field of view through which the object passes betweenthe first reflecting surface and the second reflecting surface, saidbeam of light being incident upon the first reflecting surface at anacute angle relative to a normal to the first reflecting surface, saiddistance and said angle having been selected such that said beam oflight is reflected back and forth between the first and secondreflecting surfaces so as to cross the field of view a defined number oftimes, said beam of light thus being incident on the object a pluralityof times as the object traverses the field of view, and (c) means forreflecting the beam of light exiting the reflecting cavity back alongits path and into the reflection cavity, so that the beam of light isagain reflected back and forth across the reflection cavity between thefirst reflecting surface and the second reflecting surface, traversingthe reflection cavity a further defined number of times.
 18. Theillumination system of claim 17, wherein said means comprises a thirdreflecting surface that reflects the beam of light exiting thereflecting cavity back along its path and into the cavity.